Modification Studies—Binding of Substrates and Coenzymes

Chemical Modification Studies—Binding of Substrates and Coenzymes 

The reactive residues of glutamate dehydrogenase have been probed with a variety of reagents and methods. Unless noted otherwise, the studies have been conducted with the bovine enzyme. 

Colman and Frieden (108) demonstrated in 1966 that acetylation of one amino group per subunit with acetic anhydride produces 80% inactivation. More extensive acetylation alters the degree of polymerization and certain kinetic parameters (276). Almost simultaneously, Anderson et al. (277) reported the reversible inhibition of GDH by pyridoxal 5 -phosphate and certain other aromatic aldehydes. The inhibition was attributed to formation of a Schilf base since reduction with NaBH4 results in irreversible inactivation. It was estimated that approximately one residue of -pyridoxyllysine had been formed per subunit. In 1969, Holbrook and Jeckel (278) inactivated the enzyme by reaction with a substituted maleimide and, subsequently, obtained the partial sequence of a tryptic peptide containing a modified lysine residue (Fig. 7). 

Piszkiewicz et al. (279) isolated a peptide containing e-pyridoxyllysine from a tryptic digest of GDH which had been inactivated by the procedure of Anderson et al. (277). The sequence of the peptide demonstrates that the substituted residue is Lys-126 both in the bovine (279) and, later, in the chicken enzymes (133,280). 

In the original publication, the reactive lysine was identified as residue 97. Because of the transposition already mentioned, the correct number of this residue is 126. 

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V. Chemical Modification Studies—Binding of Substrates and Coenzymes... 

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